Receptors are among the most extensively regulated cell surface components because they constitute the primary means by which cells perceive their environment. Epidermal growth factor receptors that contain intrinsic protein tyrosine kinase a activity are vectorially transported to basolateral surfaces of polarized cells and are diffusely distributed on cell surfaces. Receptors are activated by ligand binding and signaling is attenuated by occupancy-induced internalization and downregulation. Analysis of mutant EGFR indicates 3 features which distinguish internalization of these signaling receptors from that of nutrient receptors: the process is occupancy-induced, it requires activation of the intrinsic enzyme activity of the receptor and it depends on a combination of sequence codes located in the C'terminus. Intracellular trafficking is distinct because EGFR are segregated for targeting to lysosomes. We propose to use the EGFR to define mechanisms which carry out each of these processes that determine EGFR concentration and cell responses. A series of mutant EGFR will be constructed to precisely identify sequence codes that specify high affinity internalization. Mutants will test the importance of structures formed by active sequences and the importance of side chain interactions for their function. These receptor mutants will be used to define determinants of vectorial targeting to basolateral membranes-both sequence codes and tyrosine kinase activity. A second series of kinase-active, internalization competent EGFR mutants will be used to define sequences that specify endosomal sorting to lysosomes. Identified sorting sequences will also be analyzed in the context of C' terminally truncated EGFR that lack kinase-active domains. We will constitute endocytosis and sorting in vitro to investigate mechanisms through which EGFR sequence and tyrosine kinase activity operate in these processes. Proteins that interact with ligand-activated EGFR via endocytic codes and are tyrosine phosphorylated will be characterized. Proteins that interact with EGFR via lysosomal targeting codes will also be characterized. Mechanisms defined for EGFR are proposed to operate for the extended receptor tyrosine kinase gene family.